Bicarbonate-Dependent Secretion and Proteolytic Processing of Recombinant Myocilin

Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc.) affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

肌纤蛋白（Myocilin）是一种功能尚不明确的细胞外糖蛋白。该蛋白的突变与青光眼密切相关——青光眼是一类以进行性、不可逆性视力丧失为特征的视神经病变，常伴随眼压升高。本团队此前的研究证实，重组肌纤蛋白会被钙蛋白酶II（calpain II）进行细胞内蛋白水解加工：该酶切割该蛋白的中央区域，释放一个N端片段与一个C端片段。青光眼相关突变会削弱肌纤蛋白的切割过程，且该过程被认为参与眼压的调控。为筛选调控重组肌纤蛋白蛋白水解加工的潜在因素，本团队采用细胞模型，分析了多种培养基参数（包括培养时长、细胞密度、pH值、碳酸氢盐浓度等）对细胞外C端片段含量的影响。研究发现，伴随培养基酸化的细胞外碳酸氢盐耗竭，会导致全长重组肌纤蛋白发生可逆的细胞内蓄积，并增强其细胞内蛋白水解加工，使细胞外C端片段的占比升高。本研究同时证实，肌纤蛋白的细胞内蓄积依赖于其N端区域。上述数据表明，房水（aqueous humor）中的碳酸氢盐水平变化，同样可能调控眼部组织中肌纤蛋白的分泌与切割过程。